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Author Notes:

E-mail: nseyfri@emory.edu

Conceived and designed the experiments: EBD NTS.

Performed the experiments: EBD YMG NTS CF.

Analyzed the data: EBD PX DMD WR.

Contributed reagents/materials/analysis tools: DMD JJL AIL JP GJB.

Wrote the paper: EBD NTS CF WR.

We are grateful to Malu Tansey for critical analysis of biological pathways and to Jeremy Herskowitz for critical comments.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Subjects:

Research Funding:

This research project was supported in part by the Proteomics and Confocal Microscopy Core Facilities of the Emory Neuroscience NINDS Core Facilities grant, P30NS055077.

E.B.D. was supported by a National Institute on Aging grant F32AG038259 and National Institutes of Health T32 grant for translational neuroscience, NS-007480.

N.T.S was supported by a National Institute on Aging grant F32AG032848-02.

W.R. was supported by Muscular Dystrophy Association grant MDA173851.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Coaggregation of RNA-Binding Proteins in a Model of TDP-43 Proteinopathy with Selective RGG Motif Methylation and a Role for RRM1 Ubiquitination

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Journal Title:

PLoS ONE

Volume:

Volume 7, Number 6

Publisher:

, Pages e38658-e38658

Type of Work:

Article | Final Publisher PDF

Abstract:

TAR DNA-binding protein 43 (TDP-43) is a major component within ubiquitin-positive inclusions of a number of neurodegenerative diseases that increasingly are considered as TDP-43 proteinopathies. Identities of other inclusion proteins associated with TDP-43 aggregation remain poorly defined. In this study, we identify and quantitate 35 co-aggregating proteins in the detergent-resistant fraction of HEK-293 cells in which TDP-43 or a particularly aggregate prone variant, TDP-S6, were enriched following overexpression, using stable isotope-labeled (SILAC) internal standards and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We also searched for differential post-translational modification (PTM) sites of ubiquitination. Four sites of ubiquitin conjugation to TDP-43 or TDP-S6 were confirmed by dialkylated GST-TDP-43 external reference peptides, occurring on or near RNA binding motif (RRM) 1. RRM-containing proteins co-enriched in cytoplasmic granular structures in HEK-293 cells and primary motor neurons with insoluble TDP-S6, including cytoplasmic stress granule associated proteins G3BP, PABPC1, and eIF4A1. Proteomic evidence for TDP-43 co-aggregation with paraspeckle markers RBM14, PSF and NonO was also validated by western blot and by immunocytochemistry in HEK-293 cells. An increase in peptides from methylated arginine-glycine-glycine (RGG) RNA-binding motifs of FUS/TLS and hnRNPs was found in the detergent-insoluble fraction of TDP-overexpressing cells. Finally, TDP-43 and TDP-S6 detergent-insoluble species were reduced by mutagenesis of the identified ubiquitination sites, even following oxidative or proteolytic stress. Together, these findings define some of the aggregation partners of TDP-43, and suggest that TDP-43 ubiquitination influences TDP-43 oligomerization.

Copyright information:

© Dammer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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